This proposal concerns the products of the genes of polyoma virus required both for virus growth and malignant cell transformation. We shall seek to define the role of post-synthetic in vivo modifications such as phosphorylation in regulating the function of these viral gene products. The approach will be both biochemical and genetic. The goal will be to define specific modification sites, to prepare mutants that lack individual sites, and to compare the mutant and wild type proteins from both a biological and a biochemical point of view. Specific aims include: 1) identification of the sites of in vivo phosphorylation of large T and middle T antigen, 2) preparation of immunologic reagents that can be used to detect each modification site and can be used to isolate denatured T antigens, 3) analysis of phosphorylation of large T and middle T in different cells and in different subcellular locations, 4) creation of viral mutants which lack individual modification sites using techniques of site specific mutagenesis, and 5) comparison of the mutant and wild type proteins in terms of both biological function (growth and transformation) and biochemical properties (enzymatic activities, DNA-binding, membrane localization).